Cardiovascular progenitor cells (CPCs) hold tremendous therapeutic potential for cardiac regenerative medicine due to their unique ability to expand and to differentiate into various heart cell types. However, to take advantage of regenerative therapy, we need to understand the mechanisms underlying the self-renewal and lineage-specific differentiation of CPCs. The current proposal focuses on elucidating a novel role of Notch signaling in CPC maintenance and differentiation. Notch is an evolutionarily conserved transmembrane protein that plays critical roles in numerous cell-fate decisions. Canonical Notch signaling is initiated by binding of extracellular ligands to Notch. This leads to intracellular cleavage and translocation of Notch into the nucleus where it binds to the transcriptional mediator RBP-J for gene activation. I demonstrated that Notch1-deficient CPCs expand dramatically with increased proliferation, similar to the phenotype of CPCs stimulated with active 2-Catenin. This phenotype is not observed in CPCs deficient for RBP-J, suggesting a non-canonical role of Notch. Notch1-deficiency significantly increased 2-Catenin signaling. This increase was not mediated by upregulation of 2-Catenin mRNA but rather by accumulation of active 2-Catenin protein, suggesting post- translational regulation. Intriguingly, the Notch regulation of 2-Catenin protein did not require classical ligand- dependent membrane cleavage of Notch or the 2-Catenin directed proteasomal degradation, but it did require endocytic proteins that traffic membranous Notch to the lysosome. Moreover, membrane-bound Notch, conventionally considered biologically inert, physically associated with active 2-Catenin and inhibited accumulation of active 2-Catenin protein. These findings reveal a previously undescribed role of membrane Notch in regulating active 2-Catenin protein levels and set the stage for a mechanistic exploration of this role in the maintenance and differentiation of CPCs. I propose to test the hypothesis that Notch antagonizes CPC self-renewal/expansion by lysosomal degradation of active 2-Catenin in a ligand/transcription-independent fashion. The specific aims of this proposal are (1) To determine if Notch1-deficient CPCs favor self-renewal over differentiation and if 2-Catenin is required for this effect; (2) To test whether membrane-bound Notch1 affects CPC expansion and differentiation and whether the cellular events require 2-Catenin; (3) To identify the role of lysosomal activity in the link between membrane-bound Notch and active 2-Catenin degradation. The proposed work will provide fundamental insights into the understanding of mechanisms controlling CPC self- renewal/differentiation decisions, a prerequisite for CPC-mediated cardiac regenerative therapeutics. The biology of membrane Notch is completely unexplored in the field of CPCs as well as in stem cells. Given highly conserved roles of Notch and Wnt/2-Catenin signaling in nearly all known stem/progenitor cell fate decisions, these studies will open up new avenues of research for regenerative medicine involving stem/progenitor cells. PUBLIC HEALTH RELEVANCE: Understanding the biology of the cells that develop into the heart, called multipotent cardiovascular progenitor cells (CPCs), is key to realizing the promise of future cell-mediated cardiac therapeutics. The proposed research aims to elucidate the ligand/transcription-independent role of a protein called Notch in CPC self- renewal and differentiation. Our work will provide mechanistic insights into understanding the self-renewal- differentiation processes of multipotent CPCs regulated by Notch, which will facilitate future cell-based cardiac therapeutics and open new avenues of investigation for non-canonical Notch biology.